Compositions and methods for genetic modification of plants
AssigneePioneer Hi-Bred International, Inc.
InventorsChristopher L. Baszczynski, Benjamin A. Bowen, David J. Peterson, Laura A. Tagliani
Life of this patent
Estimating the term…
Abstract
Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.
Claims28
A method to directly select transformed plant cells, said method comprising:
- a) transforming said plant cell with a transfer cassette, said transfer cassette comprises a first recombination site, a nucleotide sequence encoding a selectable marker gene not operably linked to a promoter, and a second recombination site, wherein said first and second sites are non-identical;
- b) wherein said plant genome comprises at least one expression cassette, said cassette comprises a promoter operably linked to a target site comprising a first non-identical recombination site a gene coding region and a second non-identical recombination site, wherein said first and second non-identical recombination site correspond to the flanking sites of said transfer cassette;
- c) providing a recombinase that recognizes and implements a recombination event between the first recombination site in the transfer cassette and the first recombination site in the target site, and a second recombination event between the second recombination site in the transfer cassette and the second recombination site in the target site;
- and,
- d) growing said plant cells on the appropriate selective agent to recover cells which have successfully undergone targeted integration of the transfer cassette at the target site leading to activation of expression of the selectable marker.
A method to minimize or eliminate expression resulting from random integration of DNA sequences into the genome of a plant cell comprising:
- a) stably introducing into the genome of the cell at least one expression cassette comprising a transcriptional promoter, an ATG translational start sequence and a target site;
- wherein,
- i) said target site comprises a first recombination site and a second recombination site, wherein said first and second recombination sites are non-identical;
- and,
- ii) said promoter is operably linked to the translational start sequence;
- b) introducing into said cell a transfer cassette comprising a nucleotide sequence of interest and two non-identical recombination sites, wherein,
- i) the translational start sequence of the nucleotide sequence of interest has been replaced with a recombination site corresponding to said first recombination site of the target site;
- and,
- ii) recombination with the target site results in the nucleotide sequence of interest being operably linked to the ATG translational start site;
- and,
- c) providing a recombinase that implements recombination at the recombination sites.
Dependencies are inferred from the claim text.